In insects, salt and water balance are regulated by several hormones, some of which promote fluid loss (diuretic hormones) and some of which inhibit fluid loss (antidiuretic hormones). The long-term objective of this research is to understand the interplay among these hormones and how the insects regulate their internal milieu. Because of their small size and large surface area, insects are extremely vulnerable to desiccation and studies of the endocrine control of excretion may, therefore, lead the way towards development of very potent, safe, and possibly even species-specific insecticides. The house cricket, Acheta, represents an excellent model system for the study of neuroendocrine regulation of excretion, particularly since it is the only species for which well-characterized, sensitive, directly- comparable bioassays exist for both the secretory and reabsorptive portions of the excretory system. Within the time constraints of this proposal, research will focus on the sythesis, release and transport of the neurohormones affecting diuresis. The specific aims of the proposed research are: 1) to isolate and purify all of the diuretic neuropeptides from the corpora cardiaca (CC) using high performance liquid chromatography (HPLC). 2) to identify which peptide is affecting which second messenger pathway (cAMP; calcium/calmodulin; diacylglycerol) using radioimmunoassay (cAMP), ionophores (calcium) and phorbol esters (diacylglycerol). 3) to map the distribution of the diuretic neuropeptides throughout the CNS of the cricket using HPLC. 4) to determine and quantify which peptides are present in the hemolymph (and are thus active in vivo) under varying hydration states using a combination of HPLC and bioassays. 5) to identify the release sites and quantify the amount of peptide released under varying hydration states using a combination of HPLC and electron microscopy. 6) to follow the sythesis of the diuretic peptides, and possibly their precursors, using radiolabeled amino acids and in vitro incubations of CC. Separation and identification will be done using HPLC.